Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed
throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of
partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule
amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates
reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout
partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The
estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765
partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major
component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in
such measurements.
Figure Digital PCR amplification plot (left) and panel read out (right) of HindIII-digested pIRMM69. pIRMM69 contains one HindIII restriction enzyme site outside the hmg and transgene fragments used as targets in PCR. Red boxes with white shade denote positive hits containing one or more target
DNA molecules, and white boxes with grey shade refer to no target being amplified.
Keywords Bioanalytical methods - Biochips/high throughput screening - Nucleic acids (DNA|RNA) - PCR - Clinical/biomedical analysis